Membrane Permeability Function Research Articles

A Genetic Locus in Elizabethkingia anophelis Associated with Elevated Vancomycin Resistance and Multiple Antibiotic Reduced Susceptibility.

The Gram-negative Elizabethkingia express multiple antibiotic resistance and cause severe opportunistic infections. Vancomycin is commonly used to treat Gram-positive infections and has also been used to treat Elizabethkingia infections, even though Gram-negative organisms possess a vancomycin permeability barrier. Elizabethkingia anophelis appeared relatively vancomycin-susceptible and challenge with this drug led to morphological changes indicating cell lysis. In stark contrast, vancomycin growth challenge revealed that E. anophelis populations refractory to vancomycin emerged. In addition, E. anophelis vancomycin-selected mutants arose at high frequencies and demonstrated elevated vancomycin resistance and reduced susceptibility to other antimicrobials. All mutants possessed a SNP in a gene (vsr1 = vancomycin-susceptibility regulator 1) encoding a PadR family transcriptional regulator located in the putative operon vsr1-ORF551, which is conserved in other Elizabethkingia spp as well. This is the first report linking a padR homologue (vsr1) to antimicrobial resistance in a Gram-negative organism. We provide evidence to support that vsr1 acts as a negative regulator of vsr1-ORF551 and that vsr1-ORF551 upregulation is observed in vancomycin-selected mutants. Vancomycin-selected mutants also demonstrated reduced cell length indicating that cell wall synthesis is affected. ORF551 is a membrane-spanning protein with a small phage shock protein conserved domain. We hypothesize that since vancomycin-resistance is a function of membrane permeability in Gram-negative organisms, it is likely that the antimicrobial resistance mechanism in the vancomycin-selected mutants involves altered drug permeability.

Development of novel whole-mount in situ hybridization (WISH).

We have designed a novel fluorescent PNA probe which can be rapidly introduced into the living cell without any complicated pretreatment. We could post-synthetically incorporate amino acid derivatives having membrane permeability function into the PNA probe by utilizing key compound 1 which we have already developed. We show that the PNA probe will be a candidate for a rapidly penetrating and non-degrading probe for WISH.

Anthracycline-induced inhibition of membrane permeability functions dependent on metabolic energy.

The influence of anthracyclines on membrane permeability functions has been investigated in HeLa cells by monitoring the efflux of fluorescein. Release of the fluorescent dye, dependent on the metabolic energy supply, occurs after the intracellular accumulation and enzymatic hydrolysis of the non-fluorescent substrate fluorescein diacetate (FDA). Flow cytometric evaluation of the efflux kinetics showed that adriamycin (ADR), N-trifluoroacetyladriamycin-14-valerate (AD-32) and daunorubicin (DNR) inhibited the permeability process. The degree of inhibition was dependent, though to different extent, on the intracellular concentration of each drug. An increase in the efflux rate was always observed when the cells were treated with the drugs in the presence of 20 mM glucose. Relationship of these effects with energetic metabolism was supported by the finding that ATP levels were lowered by the drugs and increased by glucose. Evaluation of the cytotoxicity induced by each drug showed that the intracellular amount necessary to inhibit cell survival by 50% was of the same order of magnitude as that which decreases to 50% membrane permeability to fluorescein. These results indicate a correspondence in the concentrations of anthracyclines required for inducing cytotoxicity and for inhibiting membrane permeability functions dependent on the metabolic energy supply.